Effects of short-term increases in personal and ambient pollutant concentrations on pulmonary and cardiovascular function: A panel study analysis of the Multicenter Ozone Study in oldEr subjects (MOSES 2).

BACKGROUND: The cardiovascular effects of ozone exposure are unclear. Using measurements from the 87 participants in the Multicenter Ozone Study of oldEr Subjects (MOSES), we examined whether personal and ambient pollutant exposures before the controlled exposure sessions would be associated with adverse changes in pulmonary and cardiovascular function. METHODS: We used mixed effects linear regression to evaluate associations between increased personal exposures and ambient pollutant concentrations in the 96 h before the pre-exposure visit, and 1) biomarkers measured at pre-exposure, and 2) changes in biomarkers from pre-to post-exposure. RESULTS: Decreases in pre-exposure forced expiratory volume in 1 s (FEV1) were associated with interquartile-range increases in concentrations of particulate matter ≤2.5 µm (PM2.5) 1 h before the pre-exposure visit (-0.022 L; 95% CI -0.037 to -0.006; p = 0.007), carbon monoxide (CO) in the prior 3 h (-0.046 L; 95% CI -0.076 to -0.016; p = 0.003), and nitrogen dioxide (NO2) in the prior 72 h (-0.030 L; 95% CI -0.052 to -0.008; p = 0.007). From pre-to post-exposure, increases in FEV1 were marginally significantly associated with increases in personal ozone exposure (0.010 L; 95% CI 0.004 to 0.026; p = 0.010), and ambient PM2.5 and CO at all lag times. Ambient ozone concentrations in the prior 96 h were associated with both decreased pre-exposure high frequency (HF) heart rate variability (HRV) and increases in HF HRV from pre-to post-exposure. CONCLUSIONS: We observed associations between increased ambient PM2.5, NO2, and CO levels and reduced pulmonary function, and increased ambient ozone concentrations and reduced HRV. Pulmonary function and HRV increased across the exposure sessions in association with these same pollutant increases, suggesting a "recovery" during the exposure sessions. These findings support an association between short term increases in ambient PM2.5, NO2, and CO and decreased pulmonary function, and increased ambient ozone and decreased HRV.

Multicenter Ozone Study in oldEr Subjects (MOSES): Part 2. Effects of Personal and Ambient Concentrations of Ozone and Other Pollutants on Cardiovascular and Pulmonary Function.

INTRODUCTION: The Multicenter Ozone Study of oldEr Subjects (MOSES) was a multi-center study evaluating whether short-term controlled exposure of older, healthy individuals to low levels of ozone (O3) induced acute changes in cardiovascular biomarkers. In MOSES Part 1 (MOSES 1), controlled O3 exposure caused concentration-related reductions in lung function with evidence of airway inflammation and injury, but without convincing evidence of effects on cardiovascular function. However, subjects' prior exposures to indoor and outdoor air pollution in the few hours and days before each MOSES controlled O3 exposure may have independently affected the study biomarkers and/or modified biomarker responses to the MOSES controlled O3 exposures. METHODS: MOSES 1 was conducted at three clinical centers (University of California San Francisco, University of North Carolina, and University of Rochester Medical Center) and included healthy volunteers 55 to 70 years of age. Consented participants who successfully completed the screening and training sessions were enrolled in the study. All three clinical centers adhered to common standard operating procedures and used common tracking and data forms. Each subject was scheduled to participate in a total of 11 visits: screening visit, training visit, and three sets of exposure visits consisting of the pre-exposure day, the exposure day, and the post-exposure day. After completing the pre-exposure day, subjects spent the night in a nearby hotel. On exposure days, the subjects were exposed for 3 hours in random order to 0 ppb O3 (clean air), 70 ppb O3, and 120 ppm O3. During the exposure period the subjects alternated between 15 minutes of moderate exercise and 15 minutes of rest. A suite of cardiovascular and pulmonary endpoints was measured on the day before, the day of, and up to 22 hours after each exposure.In MOSES Part 2 (MOSES 2), we used a longitudinal panel study design, cardiopulmonary biomarker data from MOSES 1, passive cumulative personal exposure samples (PES) of O3 and nitrogen dioxide (NO2) in the 72 hours before the pre-exposure visit, and hourly ambient air pollution and weather measurements in the 96 hours before the pre-exposure visit. We used mixed-effects linear regression and evaluated whether PES O3 and NO2 and these ambient pollutant concentrations in the 96 hours before the pre-exposure visit confounded the MOSES 1 controlled O3 exposure effects on the pre- to post-exposure biomarker changes (Aim 1), whether they modified these pre- to post-exposure biomarker responses to the controlled O3 exposures (Aim 2), whether they were associated with changes in biomarkers measured at the pre-exposure visit or morning of the exposure session (Aim 3), and whether they were associated with differences in the pre- to post-exposure biomarker changes independently of the controlled O3 exposures (Aim 4). RESULTS: Ambient pollutant concentrations at each site were low and were regularly below the National Ambient Air Quality Standard levels. In Aim 1, the controlled O3 exposure effects on the pre- to post-exposure biomarker differences were little changed when PES or ambient pollutant concentrations in the previous 96 hours were included in the model, suggesting these were not confounders of the controlled O3 exposure/biomarker difference associations. In Aim 2, effects of MOSES controlled O3 exposures on forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC) were modified by ambient NO2 and carbon monoxide (CO), and PES NO2, with reductions in FEV1 and FVC observed only when these concentrations were "Medium" or "High" in the 72 hours before the pre-exposure visit. There was no such effect modification of the effect of controlled O3 exposure on any other cardiopulmonary biomarker.As hypothesized for Aim 3, increased ambient O3 concentrations were associated with decreased pre-exposure heart rate variability (HRV). For example, high frequency (HF) HRV decreased in association with increased ambient O3 concentrations in the 96 hours before the pre-exposure visit (-0.460 ln[ms2]; 95% CI, -0.743 to -0.177 for each 10.35-ppb increase in O3; P = 0.002). However, in Aim 4 these increases in ambient O3 were also associated with increases in HF and low frequency (LF) HRV from pre- to post-exposure, likely reflecting a "recovery" of HRV during the MOSES O3 exposure sessions. Similar patterns across Aims 3 and 4 were observed for LF (the other primary HRV marker), and standard deviation of normal-to-normal sinus beat intervals (SDNN) and root mean square of successive differences in normal-to-normal sinus beat intervals (RMSSD) (secondary HRV markers).Similar Aim 3 and Aim 4 patterns were observed for FEV1 and FVC in association with increases in ambient PM with an aerodynamic diameter ≤ 2.5 µm (PM2.5), CO, and NO2 in the 96 hours before the pre-exposure visit. For Aim 3, small decreases in pre-exposure FEV1 were significantly associated with interquartile range (IQR) increases in PM2.5 concentrations in the 1 hour before the pre-exposure visit (-0.022 L; 95% CI, -0.037 to -0.006; P = 0.007), CO in the 3 hours before the pre-exposure visit (-0.046 L; 95% CI, -0.076 to -0.016; P = 0.003), and NO2 in the 72 hours before the pre-exposure visit (-0.030 L; 95% CI, -0.052 to -0.008; P = 0.007). However, FEV1 was not associated with ambient O3 or sulfur dioxide (SO2), or PES O3 or NO2 (Aim 3). For Aim 4, increased FEV1 across the exposure session (post-exposure minus pre-exposure) was marginally significantly associated with each 4.1-ppb increase in PES O3 concentration (0.010 L; 95% CI, 0.004 to 0.026; P = 0.010), as well as ambient PM2.5 and CO at all lag times. FVC showed similar associations, with patterns of decreased pre-exposure FVC associated with increased PM2.5, CO, and NO2 at most lag times, and increased FVC across the exposure session also associated with increased concentrations of the same pollutants, reflecting a similar recovery. However, increased pollutant concentrations were not associated with adverse changes in pre-exposure levels or pre- to post-exposure changes in biomarkers of cardiac repolarization, ST segment, vascular function, nitrotyrosine as a measure of oxidative stress, prothrombotic state, systemic inflammation, lung injury, or sputum polymorphonuclear leukocyte (PMN) percentage as a measure of airway inflammation. CONCLUSIONS: Our previous MOSES 1 findings of controlled O3 exposure effects on pulmonary function, but not on any cardiovascular biomarker, were not confounded by ambient or personal O3 or other pollutant exposures in the 96 and 72 hours before the pre-exposure visit. Further, these MOSES 1 O3 effects were generally not modified, blunted, or lessened by these same ambient and personal pollutant exposures. However, the reductions in markers of pulmonary function by the MOSES 1 controlled O3 exposure were modified by ambient NO2 and CO, and PES NO2, with reductions observed only when these pollutant concentrations were elevated in the few hours and days before the pre-exposure visit. Increased ambient O3 concentrations were associated with reduced HRV, with "recovery" during exposure visits. Increased ambient PM2.5, NO2, and CO were associated with reduced pulmonary function, independent of the MOSES-controlled O3 exposures. Increased pollutant concentrations were not associated with pre-exposure or pre- to post-exposure changes in other cardiopulmonary biomarkers. Future controlled exposure studies should consider the effect of ambient pollutants on pre-exposure biomarker levels and whether ambient pollutants modify any health response to a controlled pollutant exposure.

Multicenter Ozone Study in oldEr Subjects (MOSES): Part 1. Effects of Exposure to Low Concentrations of Ozone on Respiratory and Cardiovascular Outcomes.

INTRODUCTION: Exposure to air pollution is a well-established risk factor for cardiovascular morbidity and mortality. Most of the evidence supporting an association between air pollution and adverse cardiovascular effects involves exposure to particulate matter (PM). To date, little attention has been paid to acute cardiovascular responses to ozone, in part due to the notion that ozone causes primarily local effects on lung function, which are the basis for the current ozone National Ambient Air Quality Standards (NAAQS). There is evidence from a few epidemiological studies of adverse health effects of chronic exposure to ambient ozone, including increased risk of mortality from cardiovascular disease. However, in contrast to the well-established association between ambient ozone and various nonfatal adverse respiratory effects, the observational evidence for impacts of acute (previous few days) increases in ambient ozone levels on total cardiovascular mortality and morbidity is mixed.Ozone is a prototypic oxidant gas that reacts with constituents of the respiratory tract lining fluid to generate reactive oxygen species (ROS) that can overwhelm antioxidant defenses and cause local oxidative stress. Pathways by which ozone could cause cardiovascular dysfunction include alterations in autonomic balance, systemic inflammation, and oxidative stress. These initial responses could lead ultimately to arrhythmias, endothelial dysfunction, acute arterial vasoconstriction, and procoagulant activity. Individuals with impaired antioxidant defenses, such as those with the null variant of glutathione S-transferase mu 1 (GSTM1), may be at increased risk for acute health effects.The Multicenter Ozone Study in oldEr Subjects (MOSES) was a controlled human exposure study designed to evaluate whether short-term exposure of older, healthy individuals to ambient levels of ozone induces acute cardiovascular responses. The study was designed to test the a priori hypothesis that short-term exposure to ambient levels of ozone would induce acute cardiovascular responses through the following mechanisms: autonomic imbalance, systemic inflammation, and development of a prothrombotic vascular state. We also postulated a priori the confirmatory hypothesis that exposure to ozone would induce airway inflammation, lung injury, and lung function decrements. Finally, we postulated the secondary hypotheses that ozone-induced acute cardiovascular responses would be associated with: (a) increased systemic oxidative stress and lung effects, and (b) the GSTM1-null genotype. METHODS: The study was conducted at three clinical centers with a separate Data Coordinating and Analysis Center (DCAC) using a common protocol. All procedures were approved by the institutional review boards (IRBs) of the participating centers. Healthy volunteers 55 to 70 years of age were recruited. Consented participants who successfully completed the screening and training sessions were enrolled in the study. All three clinical centers adhered to common standard operating procedures (SOPs) and used common tracking and data forms. Each subject was scheduled to participate in a total of 11 visits: screening visit, training visit, and three sets of exposure visits, each consisting of the pre-exposure day, the exposure day, and the post-exposure day. The subjects spent the night in a nearby hotel the night of the pre-exposure day.On exposure days, the subjects were exposed for three hours in random order to 0 ppb ozone (clean air), 70 ppb ozone, and 120 ppm ozone, alternating 15 minutes of moderate exercise with 15 minutes of rest. A suite of cardiovascular and pulmonary endpoints was measured on the day before, the day of, and up to 22 hours after, each exposure. The endpoints included: (1) electrocardiographic changes (continuous Holter monitoring: heart rate variability [HRV], repolarization, and arrhythmia); (2) markers of inflammation and oxidative stress (C-reactive protein [CRP], interleukin-6 [IL-6], 8-isoprostane, nitrotyrosine, and P-selectin); (3) vascular function measures (blood pressure [BP], flow-mediated dilatation [FMD] of the brachial artery, and endothelin-1 [ET-1]; (4) venous blood markers of platelet activation, thrombosis, and microparticle-associated tissue factor activity (MP-TFA); (5) pulmonary function (spirometry); (6) markers of airway epithelial cell injury (increases in plasma club cell protein 16 [CC16] and sputum total protein); and (7) markers of lung inflammation in sputum (polymorphonuclear leukocytes [PMN], IL-6, interleukin-8 [IL-8], and tumor necrosis factor-alpha [TNF-α]). Sputum was collected only at 22 hours after exposure.The analyses of the continuous electrocardiographic monitoring, the brachial artery ultrasound (BAU) images, and the blood and sputum samples were carried out by core laboratories. The results of all analyses were submitted directly to the DCAC.The variables analyzed in the statistical models were represented as changes from pre-exposure to post-exposure (post-exposure minus pre-exposure). Mixed-effect linear models were used to evaluate the impact of exposure to ozone on the prespecified primary and secondary continuous outcomes. Site and time (when multiple measurements were taken) were controlled for in the models. Three separate interaction models were constructed for each outcome: ozone concentration by subject sex; ozone concentration by subject age; and ozone concentration by subject GSTM1 status (null or sufficient). Because of the issue of multiple comparisons, the statistical significance threshold was set a priori at P < 0.01. RESULTS: Subject recruitment started in June 2012, and the first subject was randomized on July 25, 2012. Subject recruitment ended on December 31, 2014, and testing of all subjects was completed by April 30, 2015. A total of 87 subjects completed all three exposures. The mean age was 59.9 ± 4.5 years, 60% of the subjects were female, 88% were white, and 57% were GSTM1 null. Mean baseline body mass index (BMI), BP, cholesterol (total and low-density lipoprotein), and lung function were all within the normal range.We found no significant effects of ozone exposure on any of the primary or secondary endpoints for autonomic function, repolarization, ST segment change, or arrhythmia. Ozone exposure also did not cause significant changes in the primary endpoints for systemic inflammation (CRP) and vascular function (systolic blood pressure [SBP] and FMD) or secondary endpoints for systemic inflammation and oxidative stress (IL-6, P-selectin, and 8-isoprostane). Ozone did cause changes in two secondary endpoints: a significant increase in plasma ET-1 (P = 0.008) and a marginally significant decrease in nitrotyrosine (P = 0.017). Lastly, ozone exposure did not affect the primary prothrombotic endpoints (MP-TFA and monocyte-platelet conjugate count) or any secondary markers of prothrombotic vascular status (platelet activation, circulating microparticles [MPs], von Willebrand factor [vWF], or fibrinogen.).Although our hypothesis focused on possible acute cardiovascular effects of exposure to low levels of ozone, we recognized that the initial effects of inhaled ozone involve the lower airways. Therefore, we looked for: (a) changes in lung function, which are known to occur during exposure to ozone and are maximal at the end of exposure; and (b) markers of airway injury and inflammation. We found an increase in forced vital capacity (FVC) and forced expiratory volume in 1 second (FEV1) after exposure to 0 ppb ozone, likely due to the effects of exercise. The FEV1 increased significantly 15 minutes after 0 ppb exposure (85 mL; 95% confidence interval [CI], 64 to 106; P < 0.001), and remained significantly increased from pre-exposure at 22 hours (45 mL; 95% CI, 26 to 64; P < 0.001). The increase in FVC followed a similar pattern. The increase in FEV1 and FVC were attenuated in a dose-response manner by exposure to 70 and 120 ppb ozone. We also observed a significant ozone-induced increase in the percentage of sputum PMN 22 hours after exposure at 120 ppb compared to 0 ppb exposure (P = 0.003). Plasma CC16 also increased significantly after exposure to 120 ppb (P < 0.001). Sputum IL-6, IL-8, and TNF-α concentrations were not significantly different after ozone exposure. We found no significant interactions with sex, age, or GSTM1 status regarding the effect of ozone on lung function, percentage of sputum PMN, or plasma CC16. CONCLUSIONS: In this multicenter clinical study of older healthy subjects, ozone exposure caused concentration-related reductions in lung function and presented evidence for airway inflammation and injury. However, there was no convincing evidence for effects on cardiovascular function. Blood levels of the potent vasoconstrictor, ET-1, increased with ozone exposure (with marginal statistical significance), but there were no effects on BP, FMD, or other markers of vascular function. Blood levels of nitrotyrosine decreased with ozone exposure, the opposite of our hypothesis. Our study does not support acute cardiovascular effects of low-level ozone exposure in healthy older subjects. Inclusion of only healthy older individuals is a major limitation, which may affect the generalizability of our findings. We cannot exclude the possibility of effects with higher ozone exposure concentrations or more prolonged exposure, or the possibility that subjects with underlying vascular disease, such as hypertension or diabetes, would show effects under these conditions.

Inhibition of protein tyrosine phosphatase activity mediates epidermal growth factor receptor signaling in human airway epithelial cells exposed to Zn2+.

Epidemiological studies have implicated zinc (Zn2+) in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to metal-laden PM inhibits protein tyrosine phosphatase (PTP) activity in human primary bronchial epithelial cells (HAEC) and leads to Src-dependent activation of EGFR signaling in B82 and A431 cells. In order to elucidate the mechanism of Zn2+-induced EGFR activation in HAEC, we treated HAEC with 500 microM ZnSO4 for 5-20 min and measured the state of activation of EGFR, c-Src and PTPs. Western blots revealed that exposure to Zn2+ results in increased phosphorylation at both trans- and autophosphorylation sites in the EGFR. Zn2+-mediated EGFR phosphorylation did not require ligand binding and was ablated by the EGFR kinase inhibitor PD153035, but not by the Src kinase inhibitor PP2. Src activity was inhibited by Zn2+ treatment of HAEC, consistent with Src-independent EGFR transactivation in HAEC exposed to Zn2+. The rate of exogenous EGFR dephosphorylation in lysates of HAEC exposed to Zn2+ or V4+ was significantly diminished. Moreover, exposure of HAEC to Zn2+ also resulted in a significant impairment of dephosphorylation of endogenous EGFR. These data show that Zn2+-induced activation of EGFR in HAEC involves a loss of PTP activities whose function is to dephosphorylate EGFR in opposition to baseline EGFR kinase activity. These findings also suggest that there are marked cell-type-specific differences in the mechanism of EGFR activation induced by Zn2+ exposure.

Effect of antioxidant supplementation on ozone-induced lung injury in human subjects.

To determine whether antioxidants can influence human susceptibility to ozone (O(3))-induced changes in lung function and airway inflammation, we placed 31 healthy nonsmoking adults (18 to 35 yr old) on a diet low in ascorbate for 3 wk. At 1 wk, subjects were exposed to filtered air for 2 h while exercising (20 L/min/m(2)), and then underwent bronchoalveolar lavage (BAL) and were randomly assigned to receive either a placebo or 250 mg of vitamin C, 50 IU of alpha-tocopherol, and 12 oz of vegetable cocktail daily for 2 wk. Subjects were then exposed to 0.4 ppm O(3) for 2 h and underwent a second BAL. On the day of the O(3) exposure, supplemented subjects were found to have significantly increased levels of plasma ascorbate, tocopherols, and carotenoids as compared with those of the placebo group. Pulmonary function testing showed that O(3)-induced reductions in FEV(1) and FVC were 30% and 24% smaller, respectively, in the supplemented cohort. In contrast, the inflammatory response to O(3) inhalation, as represented by the percent neutrophils and the concentration of interleukin-6 recovered in the BAL fluid at 1 h after O(3) exposure was not different for the two groups. These data suggest that dietary antioxidants protect against O(3)-induced pulmonary function decrements in humans.

Cellular and biochemical response of the human lung after intrapulmonary instillation of ferric oxide particles.

Bronchoalveolar lavage (BAL) was used to sample lung cells and biochemical components in the lung air spaces at various times from 1 to 91 d after intrapulmonary instillation of 2.6 microm-diameter iron oxide particles in human subjects. The instillation of particles induced transient acute inflammation during the first day post instillation (PI), characterized by increased numbers of neutrophils and alveolar macrophages as well as increased amounts of protein, lactate dehydrogenase, and interleukin-8 in BAL fluids. This response was subclinical and was resolved within 4 d PI. A similar dose-dependent response was seen in rats 1 d after intratracheal instillation of the same particles. The particles contained small amounts of soluble iron (240 ng/mg) and possessed the capacity to catalyze oxidant generation in vitro. Our findings indicate that the acute inflammation after particle exposure may, at least partially, be the result of oxidant generation catalyzed by the presence of residual amounts of ferric ion, ferric hydroxides, or oxyhydroxides associated with the particles. These findings may have relevance to the acute health effects associated with increased levels of ambient particulate air pollutants.

Discordant organ laterality in monozygotic twins with primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a genetic disease characterized by abnormal ciliary structure and function, impaired mucociliary clearance, and chronic middle ear, sinus, and lung disease. PCD is associated with situs inversus in approximately 50% of the patients. One proposed explanation for this relationship is that normal ciliary function plays a role in normal organ orientation, whereas organ orientation in PCD is a random event because of dysfunctional cilia in early embryonic development. Another hypothesis for the association between PCD and situs inversus is that mutated genes in PCD not only cause defective cilia, but are also linked to the control of organ laterality, such that abnormalities in this molecular pathway result in random left-right asymmetry. We report on a set of monozygotic twin women with PCD. In both patients, deficiency of the inner dynein arms was noted on ciliary ultrastructural analysis, associated with a clinical syndrome of bronchiectasis, chronic sinusitis, and middle ear disease. One of the twins has situs solitus, the other has situs inversus totalis. DNA analysis confirmed that the twins are monozygotic. This is consistent with the hypothesis that situs inversus occurring in patients with primary ciliary dyskinesia is a random but "complete" event in the fetal development of patients with PCD.

Nociceptive mechanisms modulate ozone-induced human lung function decrements.

We have previously suggested that ozone (O3)-induced pain-related symptoms and inhibition of maximal inspiration are due to stimulation of airway C fibers (M. J. Hazucha, D. V. Bates, and P. A. Bromberg. J. Appl. Physiol. 67: 1535-1541, 1989). If this were so, pain suppression or inhibition by opioid-receptor agonists should partially or fully reverse O3-induced symptomatic and lung functional responses. The objectives of this study were to determine whether O3-induced pain limits maximal inspiration and whether endogenous opioids contribute to modulation of the effects of inhaled O3 on lung function. The participants in this double-blind crossover study were healthy volunteers (18-59 yr) known to be "weak" (WR; n = 20) and "strong" O3 responders (SR; n = 42). They underwent either two 2-h exposures to air or two 2-h exposures to 0. 42 parts/million O3 with moderate intermittent exercise. Immediately after post-O3 spirometry, the WR were randomly given either naloxone (0.15 mg/kg iv) or saline, whereas SR randomly received either sufentanil (0.2 microgram/kg iv) or saline. O3 exposure significantly (P < 0.001) impaired lung function. In SR, sufentanil rapidly, although not completely, reversed both the chest pain and spirometric effects (forced expiratory volume in 1 s; P < 0.0001) compared with saline. Immediate postexposure administration of saline or naloxone had no significant effect on WR. Plasma beta-endorphin levels were not related to an individual's O3 responsiveness. Cutaneous pain variables showed a nonsignificant weak association with O3 responsiveness. These observations demonstrate that nociceptive mechanisms play a key role in modulating O3-induced inhibition of inspiration but not in causing lack of spirometric response to O3 exposure in WR.

Activation of MAPKs in human bronchial epithelial cells exposed to metals.

We have previously shown that in vitro exposure to metallic compounds enhances expression of interleukin (IL)-6, IL-8, and tumor necrosis factor-alpha in human bronchial epithelial cells. To characterize signaling pathways involved in metal-induced expression of inflammatory mediators and to identify metals that activate them, we studied the effects of As, Cr, Cu, Fe, Ni, V, and Zn on the mitogen-activated protein kinases (MAPK) extracellular receptor kinase (ERK), c-Jun NH2-terminal kinase (JNK), and P38 in BEAS cells. Noncytotoxic concentrations of As, V, and Zn induced a rapid phosphorylation of MAPK in BEAS cells. Activity assays confirmed marked activation of ERK, JNK, and P38 in BEAS cells exposed to As, V, and Zn. Cr and Cu exposure resulted in a relatively small activation of MAPK, whereas Fe and Ni did not activate MAPK under these conditions. Similarly, the transcription factors c-Jun and ATF-2, substrates of JNK and P38, respectively, were markedly phosphorylated in BEAS cells treated with As, Cr, Cu, V, and Zn. The same acute exposure to As, V, or Zn that activated MAPK was sufficient to induce a subsequent increase in IL-8 protein expression in BEAS cells. These data suggest that MAPK may mediate metal-induced expression of inflammatory proteins in human bronchial epithelial cells.

Retention and intracellular distribution of instilled iron oxide particles in human alveolar macrophages.

Bronchoalveolar lavage (BAL) was used to sample retention of particles within the alveolar macrophage (AM) compartment at various times from 1 to 91 d following intrapulmonary instillation of 2. 6-microm-diameter iron oxide (Fe2O3) particles in human subjects. Particles were cleared from the lavagable AM compartment in a biphasic pattern, with a rapid-phase clearance half-time of 0.5 d and long-term clearance half-time of 110 d, comparable to retention kinetics determined by more traditional methods. The intracellular distribution of particles within lavaged AMs was similar in bronchial and alveolar BAL fractions. AMs with high intracellular particle burdens disappeared from the lavagable phagocytic AM population disproportionately more rapidly (shorter clearance half-time) than did AMs with lower particle burdens, consistent with the occurrence of a particle redistribution phenomenon as previously described in similar studies in rats. The rates of AM disappearance from the various particle burden categories was generally slightly slower in bronchial fractions than in alveolar fractions. The instillation of particles induced a transient acute inflammatory response at 24 h postinstillation (PI), characterized by increased numbers of neutrophils and alveolar macrophages in BAL fluids. This response was subclinical and was resolved within 4 d PI.

Rodent models of cardiopulmonary disease: their potential applicability in studies of air pollutant susceptibility.

The mechanisms by which increased mortality and morbidity occur in individuals with preexistent cardiopulmonary disease following acute episodes of air pollution are unknown. Studies involving air pollution effects on animal models of human cardiopulmonary diseases are both infrequent and difficult to interpret. Such models are, however, extensively used in studies of disease pathogenesis. Primarily they comprise those developed by genetic, pharmacologic, or surgical manipulations of the cardiopulmonary system. This review attempts a comprehensive description of rodent cardiopulmonary disease models in the context of their potential application to susceptibility studies of air pollutants regardless of whether the models have been previously used for such studies. The pulmonary disease models include bronchitis, emphysema, asthma/allergy, chronic obstructive pulmonary disease, interstitial fibrosis, and infection. The models of systemic hypertension and congestive heart failure include: those derived by genetics (spontaneously hypertensive, Dahl S. renin transgenic, and other rodent models); congestive heart failure models derived by surgical manipulations; viral myocarditis; and cardiomyopathy induced by adriamycin. The characteristic pathogenic features critical to understanding the susceptibility to inhaled toxicants are described. It is anticipated that this review will provide a ready reference for the selection of appropriate rodent models of cardiopulmonary diseases and identify not only their pathobiologic similarities and/or differences to humans but also their potential usefulness in susceptibility studies.

Epithelial cell-conditioned media inhibits degranulation of the RBL-2H3 rat mast cell line.

The effect of epithelial cells on mast cell responses was investigated by examination of degranulation of the rat mast cell line RBL-2H3 after overnight culture in media conditioned by the BEAS-2B human bronchial epithelial cell line [epithelial cell-conditioned media (ECM)]. These studies indicate that BEAS-2B cells secrete an inhibitor(s) of immunoglobulin E and A-23187-mediated degranulation of the RBL-2H3 cell line. The inhibitory activities of ECM are recovered after filtration through a 3-kDa cutoff filter. Pharmacological inhibition of cyclooxygenase in the BEAS-2B cells before preparation of ECM has no effect on subsequent inhibition of mast cell degranulation by ECM. However, cycloheximide treatment of the BEAS-2B cells before the conditioning process does preclude development of mast cell inhibitor activity in ECM, suggesting that this activity depends on protein synthesis. The effects of ECM on mast cell function are reversible, demonstrating that these effects do not result from overt cytotoxicity. Finally, media conditioned by primary cultures of human respiratory epithelial cells, but not fibroblasts, influence RBL-2H3 degranulation in a manner similar to ECM, suggesting that secretion of mast cell inhibitors may be somewhat unique to epithelial cells.

Disruption of protein tyrosine phosphate homeostasis in bronchial epithelial cells exposed to oil fly ash.

Residual oil fly ash (ROFA) is a toxic air pollutant that we have previously shown induces inflammatory mediator expression in human bronchial epithelial cells. To identify intracellular signaling mechanisms activated by ROFA, we studied its effect on protein tyrosine phosphate metabolism in the human bronchial epithelial cell line BEAS. Noncytotoxic levels of ROFA induced significant dose- and time-dependent increases in protein tyrosine phosphate levels in BEAS cells. ROFA-induced increases in protein phosphotyrosines were associated with its soluble fraction and were mimicked by vanadyl [V(IV)]- and vanadate [V(V)]-containing solutions. Ferrous, ferric, and nickel (II) ion solutions failed to increase phosphotyrosine levels. Tyrosine phosphatase activity, which was known to be inhibited by vanadium ions, was markedly diminished after ROFA treatment. Tyrosine kinase activity was unaffected. We conclude that ROFA exposure induces vanadium ion-mediated inhibition of tyrosine phosphatase activity, leading to accumulation of protein phosphotyrosines in BEAS cells. These findings demonstrate that ROFA exposure disrupts protein tyrosine phosphate homeostasis in BEAS cells and suggest a possible mechanism that leads to increased synthesis of proinflammatory proteins in airway epithelial cells exposed to PM10.

Induction of prostaglandin H synthase 2 in human airway epithelial cells exposed to residual oil fly ash.

Exposure to ambient air containing respirable particulate matter at concentrations below the current National Ambient Air Quality Standard has been associated with increased rates of pulmonary-related morbidity and mortality. To identify mechanisms involved in pulmonary responses to such exposure, we studied the effects of the emission source particulate air pollutant residual oil fly ash (ROFA) on prostaglandin metabolism in cultured human airway epithelial cells. Epithelial cells exposed to ROFA for 24 hr secreted substantially increased amounts of the prostaglandin H synthase (PHS) products prostaglandins E2 and F2 alpha. The ROFA-induced increase in prostaglandin synthesis was correlated with a marked increase in PHS activity. Western blots showed that ROFA exposure induced dose-dependent increases in PHS2 protein levels. Reverse transcriptase-PCR analyses demonstrated accompanying increases in PHS2 mRNA which were evident by 2 hr of continuous exposure. In contrast, expression of PHS1 was not affected by ROFA treatment of airway epithelial cells. There were no alterations in arachidonic acid release, incorporation, or availability in ROFA-exposed cells. These data show that exposure to ROFA induces PHS2 expression, leading to increased prostaglandin synthesis in cultured airway epithelial cells. These findings suggest that prostaglandins may play a role in the toxicology of air pollution particle inhalation.

Effects of cyclo-oxygenase inhibition on ozone-induced respiratory inflammation and lung function changes.

Inhalation of O3 causes airways neutrophilic inflammation accompanied by other changes including increased levels of cyclo-oxygenase products of arachidonic acid in bronchoalveolar lavage fluid (BALF). Ozone O3 exposure also causes decreased forced vital capacity (FVC) and forced expiratory volume after 1 s (FEV(1)), associated with cough and substernal pain on inspiration, and small increases in specific airway resistance (SRAW). The spirometric decrements are substantially blunted by pretreatment with indomethacin. Since the O3-induced decrement in FVC is due to involuntary inhibition of inspiration, a role for stimulation of nociceptive respiratory tract afferents has been suggested and cyclo-oxygenase products have been hypothesized to mediate this stimulation. However, the relation (if any) between the O3-induced neutrophilic airways inflammation and decreased inspiratory capacity remains unclear. We studied the effects of pharmacologic inhibition of O3-induced spirometric changes on the inflammatory changes. Each of ten healthy men was exposed twice (5-week interval) to 0.4 ppm O3 for 2 h, including 1 h of intermittent exercise (ventilation 601*min(-1)). One-and-a-half hours prior to and midway during each exposure the subject ingested 800 mg and 200 mg, respectively, of the non-steroidal anti-inflammatory drug ibuprofen (IBU), or placebo [PLA (sucrose)], in randomized, double-blind fashion. Spirometry and body plethysmography were performed prior to drug administration, and before and after O3 exposure. Immediately following postexposure testing, fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) was performed. Neither IBU nor PLA administration changed pre-exposure lung function. O3 exposure (with PLA) caused a significant 17 percent mean decrement in FEV(1) (P <0.01) and a 56 percent increase in mean SRAW. Following IBU pretreatment, O3 exposure induced a significantly lesser mean decrement in FEV(1) (7 percent) but still a 50 percent increase in mean SRAW. IBU pretreatment significantly decreased post-O3 BAL levels of prostaglandin E2 (PGE2) by 60.4 percent (P <0.05) and thromboxane B(2) (TxB(2)) by 25.5 percent (P <0.05). Of the proteins, only interleukin-6 was significantly reduced (45 percent, P <0.05) by IBU as compared to PLA pretreatment. As expected, O3 exposure produced neutrophilia in BALF. There was, however, no effect of IBU on this finding. None of the major cell types in the BALF differed significantly between pretreatments. We found no association between post-exposure changes of BALF components and pulmonary function decrements. We conclude that IBU causes significant inhibition of O3-induced increases in respiratory tract PGE(2) and TxB(2) levels concomitant with a blunting of the spirometric response. This is consistent with the hypothesis that the products of AA metabolism mediate inhibition of inspiration. However, IBU did not alter the modest SRAW response to O3.

Ozone-induced human respiratory dysfunction and disease.

Exercising volunteers exposed in chambers to as little as 80 ppb O3 for several hours exhibit impaired lung function and irritative lower airway symptoms. Comparable changes occur among children and young adults exposed to summer smog containing O3. Intensity of the response is reproducible but varies widely among individuals. The (reversible) decrements in vital capacity are due to involuntary inhibition of deep inspiration probably mediated by nociceptive bronchial C-fibers that may be stimulated by local prostaglandin release, and can be modulated by appropriate pharmacologic agents. A second characteristic response to low O3 levels is mucosal neutrophilic inflammation probably mediated by phospholipid-derived products and by epithelial cell-derived chemokines and cytokines, but poorly correlated with lung function changes. Fluctuations in ambient O3 levels are associated with acute respiratory health effects in exposed populations but concomitant acid aerosol pollution is an important confounder. Whether irreversible impairment of lung function occurs among residents of chronically high ozone-pollution areas is debated.

Bronchoscopic determination of ozone uptake in humans.

Measurements of ozone uptake efficiency in the human respiratory tract provide critical information toward understanding ozone dose-response characteristics. We measured ozone uptake efficiency by different regions of the respiratory tract between the mouth and bronchus intermedius in 10 healthy, resting, nonsmoking male and female subjects. The distal end of a bronchoscope was sequentially positioned at the bronchus intermedius (BI), main carina (CAR), upper trachea, and above the vocal cords. Ozone concentration was measured continuously at each sight using a rapid-responding ozone analyzer. During sampling subjects breathed through a mouthpiece connected to a pneumotachograph at a paced rate of 12 breaths/min. Integration of the product of the flow and ozone concentrations during inspiration and expiration provided the ozone mass passing each anatomic location during each phase of respiration. On inspiration the uptake efficiencies of ozone by structures between the mouth and each location j (Em-j) were 0.176 +/- 0.037 (SE), 0.271 +/- 0.024, 0.355 +/- 0.030, and 0.325 +/- 0.031 for above the vocal cords, upper trachea, CAR, and BI, respectively. A significant effect of location on Em-j was found by analysis of variance (P < 0.0002). Pairwise comparisons showed that Em-j increased as the lung penetration increased except between CAR and BI, which was not significantly different.

Respiratory responses of asthmatics to ozone.

Asthmatic individuals in the general population appear to be susceptible to disease exacerbation during summertime 'smog' episodes (ambient air pollution containing other pollutants in addition to ozone). Although controlled exposure to ozone causes acute decrements in lung function, asthmatic subjects are only marginally more susceptible to these effects. Ozone exposure also causes respiratory tract inflammatory changes, both in normals and asthmatics. Recent studies suggest that ozone pre-exposure augments the responses of allergic asthmatics to nasal and inhalation challenge with specific antigen. This may offer one possible explanation for the findings of field studies.

Effect of regional circulation patterns on observed HbCO levels.

In an earlier experiment, we briefly exposed 15 young men to high levels of CO while simultaneously monitoring arterial and peripheral venous HbCO levels. The arterial HbCO levels were considerably higher than the venous levels during the CO exposure. Furthermore, great variation in the difference between arterial and venous HbCO levels was observed, with the maximal difference for each subject ranging from 2.3 to 12.1% HbCO. In the present paper, we suggest an explanation for the observed differences between arterial and venous HbCO on the basis of the regional circulation of the forearm, where both samples were taken. Because regional circulation patterns are known to vary with physical training, the differences in physical training between subjects may account for the observed variation. An expanded model was derived from the Coburn-Forster-Kane equation, which reflects the above hypothesis. Most of the parameter values for the expanded model were measured on individual subjects. Literature values were used for other parameters. Two parameters were estimated using five of the subjects and were then used in the predictions of the expanded model for the remaining subjects.

Lung function response of healthy women after sequential exposures to NO2 and O3.

Since NOx emissions bear a precursor-product relation with ambient ozone (O3) levels, the sequence of peak ambient concentrations is first nitrogen dioxide (NO2) followed later in the day by ozone (O3). We ascertained whether preliminary exposure to 0.6 parts per million (ppm) NO2 would affect the lung function response to subsequent exposure to 0.3 ppm O3. Twenty-one healthy young nonsmoking women (18 to 35 yr of age) underwent two sets of exposures on two different days separated by a minimum of 2 wk. On one day, subjects were exposed to air for 2 h followed 3 h later by a 2-h exposure to O3. On the other day, the first exposure was to NO2; order of the days was randomized. During each exposure subjects intermittently exercised, alternating 15 min of rest with 15 min of exercise (Ve approximately 40 L/min). Spirometry was performed before the first exposure and at 1-h intervals until the end of the 2-h (O3) exposure. Plethysmography measurements were made before and after NO2 and O3 exposures. Nonspecific airway reactivity (AR) was determined at least 1 wk prior to the first exposure and following each O3 exposure. AR to methacholine (MCh) was expressed as dose required to decrease FEV1 by 10% (PD10FEV1). Nitrogen dioxide exposure alone did not reduce FEV1 but did significantly enhance O3-induced spirometric changes. No significant effects were observed in plethysmography. On both exposure days, the median PD10FEV1 was significantly reduced (p < 0.05) from control PD10FEV1 (14.3 mg/ml).(ABSTRACT TRUNCATED AT 250 WORDS)